The translocation assembly module (TAM) catalyzes the assembly of bacterial outer membrane proteins in vitro

The translocation and assembly module (TAM) has been proposed to play a crucial role in the assembly of a small subset of outer membrane proteins (OMPs) in Proteobacteria based on experiments conducted in vivo using tamA and tamB mutant strains and in vitro using biophysical methods. TAM consists of an OMP (TamA) and a periplasmic protein that is anchored to the inner membrane by a single α helix (TamB). Here we examine the function of the purified E. coli complex in vitro after reconstituting it into proteoliposomes. We find that TAM catalyzes the assembly of four model OMPs nearly as well as the β-barrel assembly machine (BAM), a universal heterooligomer that contains a TamA homolog (BamA) and that catalyzes the assembly of almost all E. coli OMPs. Consistent with previous results, both TamA and TamB are required for significant TAM activity. Our study provides direct evidence that TAM can function as an independent OMP insertase and describes a new method to gain insights into TAM function.

a Methylophaga aminisulfidivorans BamA is split into two segments in the UniprotKB database.
b Because there are no species in this Proteobacterial family that has BamA, TamA and TpsB proteins annotated in the UniprotKB database, a TpsB protein identified in a different species from the same family was used.
c There are no TpsB proteins annotated in this Proteobacterial family in the UniprotKB database.

Supplementary Fig. 2 .
Purification and reconstitution of TamA and BAM. a and d SDS-PAGE analysis of fractions eluted from Ni-NTA agarose.In a, His8-TamA was purified from cells transformed with pXW49, and in d, BamABCDE-His8 was purified from cells transformed with pYG120.b and e Purified TamA or BAM was mixed with PLE liposomes.The total mixture (T) was ultracentrifuged to generate a supernatant (S), which contains free protein, and a pellet (P), which contains reconstituted protein.Samples were analyzed by SDS-PAGE and Coomassie blue staining.c and f TamA/PLE or BAM/PLE proteoliposomes were treated with trypsin to digest exposed proteins and protein segments or untreated.The reaction was stopped by adding a trypsin inhibitor c or by heating to 95 o C for 10 min f and analyzed by SDS-PAGE and Coomassie blue staining.The low molecular weight (~10 kDa) bands seen in the presence of trypsin in f have been observed previously 1 .TamA and BAM were purified and reconstituted into proteoliposomes three and five times, respectively, with similar results.

. 3 .Supplementary Fig. 4 .plotted by the EzMol server 8 .
Purification and reconstitution of TamA G271C, G574C -TamB.His8-TamA G271C, G574C -TamB was expressed in BL21-CodonPlus(DE3)-RIPL transformed with pXW50 and purified using the same protocol that was used to purify TAM.The two residues that were mutated to cysteine are highlighted in a. b SDS-PAGE analysis of fractions eluted from Ni-NTA agarose.The two bands that migrated between 50kDa and 70kDa correspond to two TamA G271C, G574C populations that contained reduced cysteines (Cys-SH SH-Cys) or oxidized cysteines (Cys-S-S-Cys).c Purified TAM G271C, G574C was mixed with PLE liposomes.The total mixture (T) was ultracentrifuged to generate a supernatant (S), which contains free protein, and a pellet (P), which contains reconstituted protein.Samples were analyzed by SDS-PAGE and Coomassie blue staining.The data show that both oxidized and reduced forms of TamA G271C, G574C can be reconstituted into PLE.d TAM G271C, G574C -TamB PLE proteoliposomes were treated digest exposed proteins and protein segments or untreated.The reaction was stopped by adding a trypsin inhibitor and mixing half of each sample with 50 mM DTT.The samples were heated at 95° C for 10 min and analyzed by SDS-PAGE and Coomassie blue staining.DTT reduced the cyclic trypsin inhibitor to a linear molecule.The data confirm that the faster migrating band in the 50-70 kDa range is the oxidized form of TamA G271C, G574C and that only the POTRA domains of TamA G271C, G574C are exposed to digestion.The faint bands migrating at 20kDa and 70kDa in lanes 3-4 were observed in TAM/PLE samples after the addition of DTT as well.TamA G271C, G574C -TamB was purified and reconstituted into proteoliposomes twice with similar results.Experiments related to Fig.3.a The structures of OmpA (PDB 1G90 for residues 22-197; PDB 2MQE for residues 201-346), EspPΔ5' (PDB 3SLO), Ag43-β (predicted by AlphaFold), and FadL (PDB 1T16) are shown 2-7 .The models are The putative PK digestion site in OmpA, the known intra-barrel cleavage site in EspPΔ5' 9 and the putative self-cleavage sites in Ag43-β 10 are indicated.The illustration was created with BioRender.com,released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.b The experiment shown in Fig. 3c was repeated, but samples were collected only after a 60 min incubation at 30 o C, PK was not added, and the samples were not heated.The gel was expanded to show the four folded (or partially folded) forms of Ag43-β more clearly.c Part of the experiment shown in Fig. 3c was repeated.Aliquots were collected from each reaction after 60 min, treated with PK, or left untreated.Samples were mixed with loading buffer and placed on ice or heated to 95° C and resolved by SDS-PAGE.The Western blot was overexposed to show the Ag43-β that was assembled by TamA more clearly.The data show that the PK-resistance patterns of Ag43-β assembled into TAM/PLE and TamA/PLE proteoliposomes were similar.d FadL was synthesized de novo in the PURExpress coupled transcription/translation system (New England BioLabs, catalog number E6800L) using the plasmid pET303::fadL26-446.The reaction was supplemented with BODIPY-FL-ε-Lys-tRNALys (Promega, catalog number L5001) to fluorescently label FadL, 2 µM SurA and 2 µM Bam/POPC proteoliposomes to analyze BAM-mediated folding of FadL as described previously 11 .The reaction was incubated at 37° C for 1 h.Samples were subjected to PK digestion or left untreated, and then heated to 95° C or left unheated.Proteins were resolved by SDS-PAGE and visualized using an Amersham Typhoon scanner at an excitation wavelength of 488 nm.Folded FadL is denoted with a square.The experiments shown in b and c were performed three times, and the experiment shown in d was performed twice with similar results.Supplementary Fig. 5. BAM co-purified with TAM does not significantly contribute to TAM-mediated OMP assembly.a As much as 200 μmol TamA (in TamA/PLE) and TAM (in ) were heated to 95° C in SDS sample buffer, and proteins were resolved by SDS-PAGE.BamA and BamD that co-purified with TamA and TAM were visualized by Western blot using the indicated antiserum.The percent of BamA and BamD in the TamA and TAM proteoliposome preparations (shown in the table) was calculated by comparing the signal in the lanes that contained 200 μmol TamA or TAM (lanes 4 and 7) to the signal generated by a 5 μmol BAM standard (lane 1).b Urea-denatured OmpA or EspPΔ5' (0.2 μM) were incubated with the indicated concentrations of BAM/PLE and/or TamA/PLE proteoliposomes at 30° C for 60 min.After adding SDS-PAGE loading buffer, OmpA samples were placed on ice, and the EspPΔ5' samples were heated to 95° C for 10 min.Proteins were resolved by SDS-PAGE and visualized by Western blot using the indicated antisera.Percent folding (or percent cleaved, which is equivalent to percent folded for EspPD5') was determined as in Fig. 4. The data show that the level of BAM that was present in TAM/PLE and TamA/PLE proteoliposomes (<0.7% of the total protein) was insufficient to account for the observed OmpA assembly.c Ureadenatured OmpA (0.2 μM) and BAM/PLE or TAM/PLE proteoliposomes (2 μM) were incubated with the indicated amount of PMSF at 30° C for 60 min.OmpA samples were mixed with SDS-PAGE loading buffer and placed on ice for 10 min.OmpA was visualized by Western blot using the anti-OmpA-C terminal peptide antiserum and quantitated as described in part b.The data confirm that the TAM that is present in TAM-PLE mediates OmpA assembly because only TAM activity, but not BAM activity, is inhibited by PMSF.Supplementary Fig. 6.TAM can fold OMPs at low concentrations in vitro.Urea-denatured OMPs (0.2 μM) were incubated with the indicated concentrations of TAM/PLE or BAM/PLE proteoliposomes at 30° C for 60 min.After adding the loading buffer, OmpA and Ag43-β samples were placed on ice, whereas EspPΔ5' samples were heated to 95° C for 10 min.Proteins were resolved by SDS-PAGE and OMP folding was assessed by Western blot using appropriate antisera.These experiments were performed twice with similar results.

. 7 .. 8 . 4 .
TAM reconstituted into PLE shows the highest folding activity in vitro.a Chemical structures of the synthetic phospholipids used in this assay.All of the phospholipids were obtained from Avanti Polar Lipids: 1-palmitoyl-2-oleoyl-glycero-3phosphocholine (POPC, catalog number 850457), dipalmitoylphosphatidylcholine (DOPC, catalog number 850375), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, catalog number 850345), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC, catalog number 850335).b Urea denatured OmpA or EspPΔ5' was incubated with 2 μM TAM proteoliposomes containing the indicated lipids at 30° C for 60 min.Unheated OmpA or heated EspPΔ5' samples were subjected to SDS-PAGE and folding was assessed by Western blot using the antisera raised against either an OmpA or EspP C-terminal peptide.These experiments were performed three times with similar results.Quantitation of the effect of chaperones on the assembly of OMPs into TAM/PLE, TamA/PLE and BAM/PLE proteoliposomes.The effect of the indicated chaperones on the assembly of OmpA a and EspPΔ5' b into TAM/PLE, TamA/PLE and BAM/PLE proteoliposomes was analyzed in three independent experiments.Percent folding (for OmpA) and percent cleaved (for EspPD5') was determined as described in the legend to Fig. Values were then normalized to the percent folding or percent cleaved observed in the control (i.e., in the absence of a chaperone) which was defined as 100%.Error bars represent the standard error of the mean.